ABSTRACTEnhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

Mentions:
To evaluate the requirement for human BCL11A enhancer sequences, we utilized HUDEP-2 cells, an immortalized human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroid precursor cell line that expresses BCL11A and predominantly β- rather than γ-globin34. We used the clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease system to generate clones of HUDEP-2 cells with deletion of the 12-kb BCL11A composite enhancer by introduction of a pair of chimeric single guide RNAs (sgRNAs). Enhancer deletion resulted in near complete loss of BCL11A expression and induction of γ-globin and HbF protein to similar levels as cells with BCL11A knockout (Fig. 1a-c), consistent with the possibility that these sequences could serve as targets for therapeutic genome editing for HbF reinduction for the β-hemoglobinopathies35. Although targeted deletions by paired double strand breaks (DSBs) may be achieved by genome editing, competing genomic outcomes include local insertion/deletion (indel) production at each cleavage site as well as inversion of the intervening segment22,23,36–38.

Mentions:
To evaluate the requirement for human BCL11A enhancer sequences, we utilized HUDEP-2 cells, an immortalized human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroid precursor cell line that expresses BCL11A and predominantly β- rather than γ-globin34. We used the clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease system to generate clones of HUDEP-2 cells with deletion of the 12-kb BCL11A composite enhancer by introduction of a pair of chimeric single guide RNAs (sgRNAs). Enhancer deletion resulted in near complete loss of BCL11A expression and induction of γ-globin and HbF protein to similar levels as cells with BCL11A knockout (Fig. 1a-c), consistent with the possibility that these sequences could serve as targets for therapeutic genome editing for HbF reinduction for the β-hemoglobinopathies35. Although targeted deletions by paired double strand breaks (DSBs) may be achieved by genome editing, competing genomic outcomes include local insertion/deletion (indel) production at each cleavage site as well as inversion of the intervening segment22,23,36–38.

ABSTRACTEnhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.